基于群體感應信號分子的抗菌劑對發(fā)光菌毒性的影響機制及預測模型
發(fā)布時間：2018-09-18 19:32
【摘要】：抗生素作為一種廣譜性抗菌藥物被廣泛應用于醫(yī)療、畜牧業(yè)、養(yǎng)殖業(yè)等領域。然而,由于抗生素濫用及含抗生素廢棄物的不合理處置,使環(huán)境中抗生素分布范圍廣度與環(huán)境范圍濃度均日益提高。由于抗生素具有低劑量、持久性等一系列暴露特征,引發(fā)的抗性基因的產(chǎn)生給人類生存環(huán)境帶來威脅,因而尋求抗生素替代品的需求日益迫切。隨著抗生素替代品的提出及研究,如群體感應抑制劑(Quorum Sensing Inhibitor,QSIs),作為抗生素替代品被廣泛研究,或與抗生素聯(lián)合運用于醫(yī)療,可以預期在環(huán)境中,QSIs將與抗生素共存。然而,針對現(xiàn)階段研究提出的群體感應抑制劑作為抗生素替代品的方案,其環(huán)境效應與環(huán)境生態(tài)風險影響等尚不明確。Vibrio fischeri作為一種生活在廣闊海洋中的發(fā)光細菌,其產(chǎn)生的細菌群體感應信號分子C6(N-(3-oxo-hexanoyl)-L-Homoserine lactone)、C8(N-octanoyl-homoserine lactone)在環(huán)境中也存在相應分布,但是目前為止,對于環(huán)境中存在的C6、C8信號分子對抗菌劑作用于發(fā)光菌時的生物效應的影響的研究仍然很少。交叉現(xiàn)象是指根據(jù)單一污染物毒性試驗數(shù)據(jù)用現(xiàn)有模型預測的劑量效應曲線與實驗獲得的污染物聯(lián)合作用劑量效應曲線之間存在交點(一個或者多個)的現(xiàn)象,可為判別化合物之間的聯(lián)合作用類型提供很好的依據(jù),近年來被廣泛研究。然而目前為止,交叉現(xiàn)象出現(xiàn)的機制尚不明確。本文以Vibrio fischeri為模式生物,磺胺類抗生素SMP、群體感應抑制劑類C30和群體感應信號分子C6、C8為研究對象,測定了C6、C8對抗生素和群體感應抑制劑單一和聯(lián)合毒性效應(0-24h)及其基于IA模型的交叉現(xiàn)象的影響。研究發(fā)現(xiàn),(1)信號分子C6對SMP、C30單一和聯(lián)合作用于發(fā)光菌時的毒性效應有抑制作用,這是因為C6可與LuxR蛋白結(jié)合,啟動luxICDABEG的表達,因而促進發(fā)光菌的發(fā)光。(2)低濃度的C8對SMP、C30單一和聯(lián)合作用于發(fā)光菌時的毒性效應為抑制,高濃度的C8對SMP、C30單一和聯(lián)合作用于發(fā)光菌時的毒性效應在0-12 h為抑制,13-24 h為促進,這是因為隨著細菌自身分泌的C6增多,外源C8會跟C6競爭結(jié)合LuxR蛋白,使C6對發(fā)光菌的刺激作用減小。(3)C6會使SMP、C30聯(lián)合作用于發(fā)光菌時出現(xiàn)交叉現(xiàn)象的濃度點增大,而C8使SMP、C30聯(lián)合作用于發(fā)光菌時出現(xiàn)交叉現(xiàn)象的濃度點減小,這是因為C6、C8屬于發(fā)光菌的兩條不同的群體感應系統(tǒng),C6屬于LuxI/Lux R系統(tǒng),C8屬于AinS/AinR系統(tǒng),在群體感應系統(tǒng)中C6比C8先發(fā)揮作用,而C8很大一部分作用是當C6濃度不足時,作為補充與LuxR蛋白結(jié)合,從而C6有使SMP+C30的聯(lián)合毒性表現(xiàn)為拮抗的傾向,而C8有使SMP+C30聯(lián)合毒性表現(xiàn)為協(xié)同的傾向。另外,面對當今污染物復雜多樣、長時間暴露的嚴重情形,開展慢性毒性的研究就變得至關重要,然而目前對于急慢性毒性預測模型的研究大都為經(jīng)驗的選擇,缺少基于急性與慢性毒性作用機制比較的理論依據(jù),因此本文采用LitR蛋白的E_(binding)與Ka/Kc擬合的方法,建立了不同化合物同一個QSAR的從急性毒性預測慢性毒性的模型。研究發(fā)現(xiàn),Lit R與靶蛋白之間有很好的相關性,這使得運用LitR的E_(binding)代替靶蛋白的E_(binding)與logKc/Ka進行擬合有了可行性。本文的研究結(jié)果不僅為抗菌劑聯(lián)合作用的生態(tài)毒理評估和環(huán)境風險評價提供理論依據(jù),而且還為交叉現(xiàn)象形成機制的進一步探索提供數(shù)據(jù)支撐和理論指導。
[Abstract]:As a broad-spectrum antibiotic, antibiotics are widely used in medical, animal husbandry, aquaculture and other fields. However, due to the abuse of antibiotics and unreasonable disposal of antibiotic-containing wastes, the distribution and concentration of antibiotics in the environment are increasing day by day. With the development of antibiotic substitutes, such as Quorum Sensing Inhibitor (QSIs), as an antibiotic substitute, it is widely studied or used in combination with antibiotics in medicine. It is expected that QSIs will coexist with antibiotics in the environment. However, the environmental effects and environmental ecological risk effects of quorum sensing inhibitors as alternatives to antibiotics are still unclear. Vibrio fischeri, a luminescent bacterium living in a vast ocean, produces a sense of bacterial community. The signal molecules C6 (N-(3-oxo-hexanoyl) -L-Homoserine lactone) and C8 (N-octanoyl-homoserine lactone) are also distributed in the environment, but so far, the effects of C6 and C8 signaling molecules on the biological effects of antimicrobial agents acting on luminescent bacteria in the environment are seldom studied. There is a phenomenon of intersection (one or more) between the dose-effect curves predicted by existing models and the dose-effect curves obtained by experiments, which can provide a good basis for identifying the types of combined action between compounds. However, the phenomenon of intersection has been widely studied in recent years. In this study, the effects of C6 and C8 on the single and combined toxicity (0-24 h) of antibiotics and quorum sensing inhibitors and their cross-phenomena based on IA model were investigated using Vibrio fischeri as model organism, sulfonamide antibiotics SMP, quorum sensing inhibitors C30 and quorum sensing signal molecules C6, C8. It was found that (1) signal molecule C6 inhibited the toxic effect of SMP and C30 on luminescent bacteria, because C6 could bind to LuxR protein and activate the expression of luxICDABEG, thus promoting the luminescence of luminescent bacteria. (2) The toxic effect of low concentration C 8 on SMP and C30 on luminescent bacteria was inhibited, and the toxic effect of high concentration C on luminescent bacteria was inhibited. The toxic effect of C8 on SMP, C30 on luminescent bacteria was inhibited at 0-12 h and promoted at 13-24 h, because exogenous C8 would compete with C6 to bind LuxR protein, and the stimulating effect of C6 on luminescent bacteria would decrease with the increase of C6 secreted by the bacteria themselves. (3) C6 would make the concentration point of cross-reaction between SMP and C30 on luminescent bacteria. C8 increased the concentration of SMP and C30, but decreased the concentration of C6 and C8, because C6 and C8 belonged to two different quorum sensing systems of luminescent bacteria. C6 belonged to LuxI/Lux R system, C8 belonged to AinS/AinR system, C6 played a role first than C8 in quorum sensing system, and C8 played a major role when the concentration of C6 was insufficient. As a complement to LuxR, C6 tends to antagonize the combined toxicity of SMP + C30, whereas C8 tends to synergize the combined toxicity of SMP + C30. In addition, chronic toxicity research is crucial in the face of the complex and diverse contaminants and the serious situation of long-term exposure. Sexual toxicity prediction models are mostly empirical choices, and lack of theoretical basis for comparing the mechanisms of acute and chronic toxicity. In this paper, the E_ (binding) and Ka/Kc fitting methods of LitR protein were used to establish a model for predicting chronic toxicity from acute toxicity of different compounds with the same QSAR. There is a good correlation between white and white, which makes it possible to use LitR E_ (binding) instead of target protein E_ (binding) to fit logKc / Ka. The results of this study not only provide a theoretical basis for ecotoxicological assessment and environmental risk assessment of antimicrobial agents, but also provide a further exploration for the formation mechanism of cross phenomena. Provide data support and theoretical guidance.
【學位授予單位】：上海海洋大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：X171.5
本文編號：2248904
[Abstract]:As a broad-spectrum antibiotic, antibiotics are widely used in medical, animal husbandry, aquaculture and other fields. However, due to the abuse of antibiotics and unreasonable disposal of antibiotic-containing wastes, the distribution and concentration of antibiotics in the environment are increasing day by day. With the development of antibiotic substitutes, such as Quorum Sensing Inhibitor (QSIs), as an antibiotic substitute, it is widely studied or used in combination with antibiotics in medicine. It is expected that QSIs will coexist with antibiotics in the environment. However, the environmental effects and environmental ecological risk effects of quorum sensing inhibitors as alternatives to antibiotics are still unclear. Vibrio fischeri, a luminescent bacterium living in a vast ocean, produces a sense of bacterial community. The signal molecules C6 (N-(3-oxo-hexanoyl) -L-Homoserine lactone) and C8 (N-octanoyl-homoserine lactone) are also distributed in the environment, but so far, the effects of C6 and C8 signaling molecules on the biological effects of antimicrobial agents acting on luminescent bacteria in the environment are seldom studied. There is a phenomenon of intersection (one or more) between the dose-effect curves predicted by existing models and the dose-effect curves obtained by experiments, which can provide a good basis for identifying the types of combined action between compounds. However, the phenomenon of intersection has been widely studied in recent years. In this study, the effects of C6 and C8 on the single and combined toxicity (0-24 h) of antibiotics and quorum sensing inhibitors and their cross-phenomena based on IA model were investigated using Vibrio fischeri as model organism, sulfonamide antibiotics SMP, quorum sensing inhibitors C30 and quorum sensing signal molecules C6, C8. It was found that (1) signal molecule C6 inhibited the toxic effect of SMP and C30 on luminescent bacteria, because C6 could bind to LuxR protein and activate the expression of luxICDABEG, thus promoting the luminescence of luminescent bacteria. (2) The toxic effect of low concentration C 8 on SMP and C30 on luminescent bacteria was inhibited, and the toxic effect of high concentration C on luminescent bacteria was inhibited. The toxic effect of C8 on SMP, C30 on luminescent bacteria was inhibited at 0-12 h and promoted at 13-24 h, because exogenous C8 would compete with C6 to bind LuxR protein, and the stimulating effect of C6 on luminescent bacteria would decrease with the increase of C6 secreted by the bacteria themselves. (3) C6 would make the concentration point of cross-reaction between SMP and C30 on luminescent bacteria. C8 increased the concentration of SMP and C30, but decreased the concentration of C6 and C8, because C6 and C8 belonged to two different quorum sensing systems of luminescent bacteria. C6 belonged to LuxI/Lux R system, C8 belonged to AinS/AinR system, C6 played a role first than C8 in quorum sensing system, and C8 played a major role when the concentration of C6 was insufficient. As a complement to LuxR, C6 tends to antagonize the combined toxicity of SMP + C30, whereas C8 tends to synergize the combined toxicity of SMP + C30. In addition, chronic toxicity research is crucial in the face of the complex and diverse contaminants and the serious situation of long-term exposure. Sexual toxicity prediction models are mostly empirical choices, and lack of theoretical basis for comparing the mechanisms of acute and chronic toxicity. In this paper, the E_ (binding) and Ka/Kc fitting methods of LitR protein were used to establish a model for predicting chronic toxicity from acute toxicity of different compounds with the same QSAR. There is a good correlation between white and white, which makes it possible to use LitR E_ (binding) instead of target protein E_ (binding) to fit logKc / Ka. The results of this study not only provide a theoretical basis for ecotoxicological assessment and environmental risk assessment of antimicrobial agents, but also provide a further exploration for the formation mechanism of cross phenomena. Provide data support and theoretical guidance.
【學位授予單位】：上海海洋大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：X171.5
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